1. Field of the Invention
This invention relates to a method of testing a patient to determine a predisposition to certain cancers and certain diseases with a hereditary background.
2. Description of the Prior Art
The American Cancer Society estimates that in the United States alone, approximately one person dies from cancer every eighty seconds. It has also ben estimated that 117,000 deaths from cancer that occurred in 1978 could have been prevented by earlier diagnosis and treatment. The primary goal of public health is to prevent diseases rather than the treatment of disease.
It is well known among cancer research workers that certain factors are closely related to the instance of cancer. Among these factors are to be mentioned the history of cancer within the family, environment and personal habits such as smoking. If a predisposition of a person to cancer were known, the predisposition would permit the early diagnosis of the existence of cancer. Such a predisposition would als facilitate the possibility of early remedial treatment thereof.
Various diagnostic techniques have been employed in the past in an effort to diagnose the existence at an early stage of the development of cancer within a patient. Such prior art techniques suffered from several inherent problems. Firstly, such techniques have often proved unreliable in diagnosing the existence of cancer. Secondly, such prior art diagnostic tools have merely established the existence of cancer within the patient, but have not been able to determine the predisposition of a patient to the occurrence of a cancerous disorder. Thirdly, the cost of conducting such prior art methods of diagnosis has usually proved to be prohibitive.
In order to achieve the important goal of preventing lung cancer, certain cancers and other hereditary diseases, the object of the present invention is to identify those individuals at relatively high risk for developing this malignancy or other disease prior to detection of frank or metastatic growth. This invention is based on observations made during in vitro studies of cellular transformation, particularly of skin fibroblast cells derived from patients who are genetically susceptible to cancer. These findings revealed increased oncogenic virus transformation of cultured fibroblasts derived from such patients.
The present invention is specifically aimed at assessing a convenient, reliable, and rapid in vitro virus transformation assay as a diagnostic index to identify those persons susceptible to certain diseases such as smokers who are at increased risk for subsequent development of lung cancer.
The present invention is founded on laboratory, clinical and epidemiological observations of cancer which clearly suggest genetic as well as environmental predisposing factors in the etiology of disease. It is important to develop a diagnostic procedure that can identify smokers and non-smokers in the population who may be at increased genetic risk for developing lung cancer and possibly other neoplastic diseases. The need for such a procedure is paramount, since present diagnostic procedures and treatment of lung and other cancers, even when applied rigorously, result only in a modest enhancement of survival rate as described in Brett, G. Z.; "Earlier Diagnosis and Survival in Lung Cancer", British Medicine. 4:260-262, 1969.
In the identification of genetic conditions that predispose to cancer, a number of widely diverse studies have been conducted in attempts to identify susceptible individuals. An example of one of these approaches concerned experiments conducted to detect the sensitivity of cells with gross chromosomal aberrations followed by increased spontaneous chromosomal breakage to certain carcinogens. In these instances, the premise was to ultimately detect cytogenetically normal cells that possessed enhanced susceptibility to cancer. In measuring chromosomal breaks per radiation dose, fibroblasts and lymphocytes from Fanconi's anemia exhibited a significantly greater sensitivity to X-ray (Higuraski, M., Conen P. E.: "In Vitro Chromosomal Radiosensitivity in Fanconi's Anemia", Blood. 38:336-342, 1971). Fanconi's anemia is an autosomal recessive disease which is associated with a high incidence of chromosome aberrations and a greatly increased risk of neoplasia (Bloom G. E. Warner S., Gerald P. S., Diamond L. K.: "Chromosome Abnormalities in Constitutional Aplastic Anemia", New England Journal of Medicine. 274:8-14, 1966 and Swift M. R., Hirschhorn K.: "Fanconi's Anemia: Inherited Susceptibility to Chromosome Breakage in Various Tissues", Annals Internal Medicine. 65:496-503, 1966).
Further, cells from patients with Fanconi's anemia or Down's Syndrome (trisomy 21) showed an increased percentage of endoreduplication and tetrapoloidy in studies with the chemical carcinogen, benzpyrene (Hirschhorn K., Block-Shtacher N.: "Transformation of Genetically Abnormal Cells", In The University of Texas M.D. Anderson Hospital and Tumor Institute at Houston, 23rd Annual Symposium on Fundamental Cancer Research, 1969: Genetic Concepts and Neoplasia. Baltimore, Williams & Wilkins, 1970 pp. 191-202). Down's Syndrome is another disease associated with an abnormally high incidence of tumors (Krivit W., Good R. A.: "Simultaneous Occurrence of Mongolism and Leukemia", American Journal of Diseases of Children. 94:289-293, 1957 and Holland W. W., Doll R., Carter C. O.: "The Mortality From Leukemia and Other Cancers Among Patients with Down's Syndrome (Mongols) and Among Their Parents", British Journal of Cancer 16:177-186, 1962); the nature of the chromosome abnormality of Down's Syndrome is, however, fundamentally quite different from that of Fanconi's anemia.
The present invention seeks to identify smokers at high risk for developing lung cancer is based upon previous studies that have utilized quantitative in vitro virus transformation of human cells. The method can provide a convenient, reliable and rapid test for assessing predisposition to lung cancer in individuals in the population with particular emphasis on those who are heavy smokers. This information is critical to any Public Health program and is designed to prevent or circumvent this disease.
Genetic factors have been shown to play a dominant role in Rous sarcoma virus focus formation in chick cells and tumor formation in the chick (Payne L. N., Briggs P. M.: "Differences Between Highly Inbred Lines of Chickens in the Response to Rous Sarcoma Virus of the Chorioallantoic Membrane and of Embryonic Cells in Tissue Culture", Virology. 24:610-616, 1964 and Vogt P. K., Ishizaki R: "Reciprocal Patterns of Genetic Resistance to Avian Tumor Viruses in Two Lines of Chickens", Virology. 26:665-667, 1965). Other studies described the influence of the cell genotype on transformation of human skin fibroblasts by oncogenic viruses. These studies were guided by early evidence that persons at high risk for cancer could be identified by relative susceptibility rate to oncogenic sarcoma virus transformation of their cultured skin fibroblasts (Higuraski M., Todaro G. J.: "Viral Transformation of Cells from Persons at High Risk of Cancer ", Lancet. 1:81-82, 1969).
Inoculation of fibroblast cultures from patients with Fanconi's anemia with simian virus 40 (SV40) and an adenovirus 7-SV40 "hybrid" revealed a much greater susceptibility of these cells to transformation when compared with fibroblast strains developed from normal individuals and from individuals not associated with increased tumor incidence (Todaro G. J., Green H, Swife M. R.: "Susceptibility of Human Diploid Fibroblast Strains to Transformation by SV40 Virus", Science. 153:1252-1254, 1966). Multiple tumor biopsies from the same person led to cell strains that were very similar in their transformation susceptibility with SV40 (Todaro G. J., Aaronson S. A.: "Human Cell Strains Susceptible to Focus Formation by Human Adenovirus Type 12", Proceedings of the National Academy of Science, U.S.A. 61:1272-1278, 1968). Subsequent results showed that skin fibroblast cultures from patients with Down's Syndrome are intrinsically more susceptible to in vitro transformation by SV40 than are normal cell cultures (Todaro G. J., Martin G. M.: "Increased Susceptibility of Down's Syndrome Fibroblasts to Transformation by SV40", Proceedings of the Society of Experimental Biology and Medicine. 124:1232-1236, 1967). The human fibroblasts that were highly susceptible to transformation by SV40 were subsequently tested with human adenovirus type 12, and these cultures developed foci of altered cells with much greater frequency than did the normal human cell cultures (Todaro, Aaronson, "Human Cell Strains, etc." pp. 1272-1278). Moloney murine sarcoma virus is an RNA-containing tumor virus (Huebner R. J.: "The Murine Leukemia-Sarcoma Virus Complex", Proceedings of the National Academy of Science, U.S.A. 58:835-842, 1967) has also been demonstrated to cause transformation of human fibroblast strains derived from a variety of sources, including adult skin (Aaronson S. A., Todaro G. J.: "Transformation and Virus Growth by Murine Sarcoma Viruses in Human Cells", Nature. 225:458-459, 1970).
The Kirsten strain of murine sarcoma virus (KiMSV) from a KiMSV transformed rat cell line (Klement V., Hartley J. W., Rowe W. P., Huebner R. J.: "Recovery of a Hamster-Specific, Focus-Forming, and Sarcomagenic Virus From a `Non-Infectious` Hamster Tumor Induced by the Kirsten Mouse Sarcoma Virus", Journal of the National Cancer Institute 43:925-933, 1968), also induced morphological transformation in cultured human cells (Aaronson, S. A., Todaro G. J.: "Transformation and Virus Growth etc." pp.458-459). KiMSV is a sarcomagenic isolate from a rate-passaged murine erythroblastosis virus that shares the rat sarcoma genome with other rat sarcomas (Rhim J., Vernon J., Dug F., Huebner R. J.: "Wide Host Range of Murine Sarcoma Virus", International Journal of Cancer 12:734-741, 1973). The morphological alternations of the KiMSV tranformed foci are characterized by refractile spindle-shaped and round cells which grow on top of the monolayers and exhibit large cytoplasmic vacuoles.
This invention utilizes KiMSV in the transformation studies since it has been shown that KiMSV is more efficient than MMSV in transforming human cell cultures (Aaronson S. A., Todaro G. J.: "Transformation and Virus Growth, etc." pp. 458-459). This may well due to the KiMSV-associated xenotropic helper virus rather than to the sarcoma genome itself (Aaronson S., Weaver C.: "Characterization of Murine Sarcoma Virus (Kirsten) Transformation of Mouse and Human Cells", Journal of General Virology. 13-245-252, 1971). The change in morphology is focal and can be quantitatively evaluated by absolute virus dose required to effect transformation (Klement V., Freedman M. H., McAllister R. M., Nelson-Rees W. A., Huebner R. J.: "Differences in Susceptibility of Human Cells to Mouse Sarcoma Virus", Journal of the National Cancer Institute. 47-65-73, 1971). The susceptibility of 18 individual fibroblast strains to transformation varied more than 300-fold (ibid). In general, as were the findings cited above relative to SV40, adenovirus and MMSV, more susceptibile cell strains were found among those derived from individuals with neoplasia and genetic or chromosomal abnormalities than those derived from "normal" individuals and fetuses (ibid). In the present invention, it was noted that: (1) Tissue cultures derived from various organs from human fetuses revealed the same type of sensitivity; (2) The cultures with low sensitivity to transformation were transformed with higher titers of KiMSV than were required for susceptible cells; and (3) A high sensitivity of some apparently "normal" human cells was observed. The findings that cell strains from apparently "normal" individuals or fetuses showed high sensitivity to focus formation is of special interest. Such individuals may well represent a specific group of "high risk persons" with an enhanced predisposition to cancer.
Hereditary adenomatosis of the colon and rectum (AGR) is a disorder in which numerous polyps develop in the gastrointestinal tract (Frameni J. Jr.: "Genetic Factors", In J. Holland and E. Frei (eds.) Cancer Medicine. pp. 7-15, Philadelphia, Lea and Febinger, 1973 and Knudson A. Jr., Strong L., Anderson D.: "Heredity and Cancer in Man", In A. Steinberg and A. Bearn (eds.), Progress in Medical Genetics, 9 pp. 113-158, New York, Grune and Stratton, 1973). Within the familial proband, it is clearly established that 50 percent of the members are at 90-100 percent risk of colon cancer (Dr. R. J. Huebner, personal communication). This trait is carried by an autosomal dominant gene, although it seems probable that additional genes may pleiotropically modify its expression (Alm T., Licznerski G.: "The Intestinal Polyposes", In R. McConnel (ed.) Clinics in Gastroenterology, pp. 577-601, Philadelphia, Saunders, 1973; and Gardner E., Richards R.: "Multiple Cutaneous and Subcutaneous Lesions Occurring Simultaneously with Hereditary Polyposis and Osteomatosis", American Journal of Human Genetics. 5:139-149, 1953; And McConnel R. B.: The Genetics of Gastrointestinal Disorders. London, Oxford University Press, 1966, pp. 1-282; and Morson B., Bussey H.: "Predisposing Causes of Intestinal Cancer", in Current Problems in Surgery. Chicago, Yearbook Medical Publishers, 1970 pp. 1-50).
Recently, it has been shown that neoplasia in ACR subjects was correlated with heightened susceptibility to transformation by KiMSV of skin fibroblasts (Pfeffer L. M., Kopelovich L.: "Differential Genetic Susceptibility of Cultured Human Skin Fibroblasts to Transformation by Kirsten Murine Sarcoma, Virus", Cell. 10:313-320, 1977). In this study, there also appeared to be a correlation heightened susceptibility to virus transformation of skin fibroblasts and loss of contact inhibition and decreased serum requirement for growth. However, an asymptomatic child of an ACR subject whose skin fibroblasts did not grow in decreased serum medium was highly susceptible to KiMSV transformation. The skin fibroblasts from ACR subjects were 100 to 1000-fold more susceptible to transformation by KiMSV than were normal cells (Kopelovich L.: "Phenotypic Markers in Human Skin Fibroblasts as Possible Diagnostic Indices of Hereditary Adenomatosis of the Colon and Rectum", Cancer. 40:2534-2541, 1977). The virus transformed skin fibroblasts formed tumors in athymic mice. These findings, as well as those described above, relative to heightened susceptibility of cultured skin fibroblasts to oncogenic virus transformation, point out the possible utility of this phenomenon for use as a diagnostic index not only for individuals with latent ACR, but those who are at high risk for other forms of cancer (Pfeffer L. M., Kopelovich L.: "Differential Genetic Susceptibility etc.", pp. 313-320; and Kopelovich L.: "Hereditary Adenomatosis of the Colon and Rectum-A Model of Tumor Progression", in S. Day (ed.) 1st International Workshop on Cancer Invasion and Metastasis: Biologic Mechanism and Therapy. pp. 375-387, New York, Raven Press, 1977; and Kopelovich L., Pfeffer L., Lipkin M.: "Recent Studies on the Identification of Proliferative Abnormalities and of Oncogenic Potential of Cutaneous Cells in Individuals at Increased Risk of Colon Cancer", Seminars in Oncology, 3:369-372, 1976; and Pfeffer L., Lipkin M., Stutman O., Kopelovich L.: "Growth Abnormalities of Cultured Human Skin Fibroblasts Derived from Individuals with Hereditary Adenomatosis of the Colon and Rectum", Journal of Cell Physiology, 89-29-38, 1976; and Kopelovich, L., Conlon S., Pollack R.: "Defective Organization of Actin in Cultured Skin Fibroblasts from Patients with Inherited Adenocarcinoma", Proceedings of the National Academy of Science, U.S.A. 74:3019-3022, 1977), such as middled aged heavy smokers.
The present invention provides a method for determining the predisposition of a patient to the development of certain cancerous growths before such cancerous disorders have been estabished. Such growths are apparent in patients with lung cancer and occurs in certain familial disorders such as neurofibromatosis, an autosomal dominant disorder characterized by a very stron predisposition to malignancy.
The benefits derived from such a method are readily apparent even to the layman. If a patient is diagnosed according to the present invention to have a predispositon to cancer, several countermeasures can be used by the patient to reduce the possibility of developing cancer. Among these countermeasures, the more obvious one to be mentioned is the avoidance of the use by the patient of tobacco and excessive use of alcohol in conjunction with smoking. Additonally, if the patient so diagnosed lives in an area close to an airport, the possibility of developing cancer as a result of exposure to the combustion products of jet and rocket fuels is proportionately higher. By moving to a locally of decreased exposure, the possibility of developing cancer can be decreased thereby.
A patient who is diagnosed as having a predisposition to cancer may be working in an environment which is inducive to the occurrence of cancer. Environments of this nature are those in which the patient has close contact with asbestos.
The method of detecting a predisposition of a patient to cancer according to this invention, also provides a relatively simple, low cost tool which enables medical personnel to reliably determine whether a patient is at risk. If a patient is diagnosed as being at risk, preventative precautions can immediately be put into effect.
In addition to the advantages listed hereinbefore, the results obtained by the use of the technique of the present invention can be utilized as a basis for a gentic prenatal counseling tool. Up to the present, such counseling of prospective parents as to the possibility of their offspring having a higher than normal risk of developing cancer has proved extremely difficult. However, with the method of the present invention, a predisposition of the unborn child to certain cancers and/or certain other hereditary disorders could be determined and appropriate counsel given the parents to be.
The diagnostic technique of the present invention envisages a test kit for taking skin samples from patients and treating the same under laboratory conditions and includes an index for comparing the results obtained to such index to determine a predisposition to certain cancers and certain other hereditary disorders. Therefore, it is the primary object of this invention to provide a method of detecting a predisposition to certain cancers and certain other hereditary disorders which overcomes the aforementioned inadequacies of the prior art methods and provides an improvement which significantly contributes to the ease with which a predisposition to cancer can be detected.
Another object of the present invention is the provision of a method of testing a patient for the existence of certain cancers and certain other hereditary disorders.
A further object of the present invention is the provision of a method for testing a patient to determine a predisposition to certain cancers and certain other hereditary disorders which is relatively low in cost.
Another object of the invention is the provisionn of a method of testing the patient to determine a predisposition to certain cancers and certain other hereditary disorders wherein the method can be performed with the aid of a test kit.
Another object of the invention is to use the results of the test to provide a counseling tool relative to a wide variety of hereditary disorders such as certain prenatal hereditary disorders such as neurofibromatosis.
The foregoing has outlined some of the more pertinent objects of the present invention. These objects should be construed to be merely illustrative of some of the more prominent features and aplications of the invention. Many other beneficial results can be obtained by applying the disclosed invention in a different manner or modifying the invention within the scope of the disclosure, such as performing the diagnostic test in a matter of days, a DNA-probe prepared from Kirsten murine sarcoma virus in utilizing detection of expression of the cellular homology of the Kirsten murine sarcoma virus genome in patients' cells and/or amplification of the same gene (analogous to C. Ras). Particularly with regard to the use of the invention disclosed herein, this should not be construed as limited to a method of testing a patient to determine a predisposition to only lung cancer and neurofibromatosis, but should include a method for determining predisposition to certain other cancers, generally certain other hereditary diseases and the like.